You must be logged in to access this feature.

RELEASE of Ca2+from intracellular stores is a widespread component in several signaling pathways. 1–2 It is well known that inositol-1,4,5-tris-phosphate (IP3) triggers Ca2+release from intracellular stores 1; however, cells possess other intracellular Ca2+releasing systems, 1–3 including the so-called Ca2+-induced Ca2+release system, mediated by the ryanodine receptor–channel (RyR). 1–2 Recently it was found that the endogenous nucleotide cADP-ribose (cADPR) is a potent activator of the RyR, 1–2 and this nucleotide has been proposed to be a second messenger in several intracellular signaling pathways. 1 Biosynthesis of cADPR from β-NAD is catalyzed by adenosine diphosphate (ADP)-ribosyl cyclase, and cADPR is hydrolysis is mediated by the cADPR hydrolase to ADP-ribose (ADPR). 1

Volatile anesthetics have multiple actions on intracellular Ca2+homeostasis, 4–9 including activation of the RyR and sensitization of this channel to pharmacologic agonists such as caffeine and ryanodine. 4–9 Recently, we reported that halothane can sensitize the RyR to cADPR in sea urchin egg homogenates. 7 It has been previously shown that the cADPR system is functional in porcine smooth muscle cells. 10 In fact, in porcine airway smooth muscle cells cADPR has been shown to be a second messenger responsible for intracellular Ca2+increase induced by acetylcholine. 10 In the current study, we found that halothane potentiates the cADPR-induced Ca2+release through the RyR in porcine airway smooth muscle cells. We propose that modulation of the cADPR signaling system by halothane may be an important component of the complex effect of this volatile anesthetic on intracellular Ca2+homeostasis.

Fluo-3 was purchased from Molecular Probes (Eugene, OR); IP3, oligomycin, and antimycin were from Calbiochem (San Diego, CA). All other reagents, of the highest purity grade available, were supplied from Sigma Chemical (St. Louis, MO).

The reported experiments were repeated at least three to six times. When appropriate, data are expressed as mean ± SD. The unpaired t
test was used to evaluate statistical significance;P
values < 0.05 were considered significant.

Results and Discussion

Activation of RyR by cADPR in Tracheal Smooth Muscle Microsomes

It has been previously shown that the RyR-cADPR system is present and functional in smooth muscle cells. 10–12 Furthermore, cADPR is able to activate the RyR in tracheal smooth muscle cells. 10 Tracheal smooth muscle cell microsomes supplemented with an ATP-regenerative system sequester added Ca2+into vesicular stores in an ATP-dependent manner and release Ca2+in response to μm concentrations of cADPR (fig. 1). The cADPR-induced Ca2+release was inhibited by several inhibitors of the RyR such as spermine, ruthenium red, and the specific cADPR inhibitor 8-Br-cADPR (fig. 1). 13 However, Ca2+release induced by cADPR was not inhibited by 1 mg/ml heparin, a specific antagonist of the IP3channel. 13 These observations confirmed the evidence that cADPR activates Ca2+release through the RyR in tracheal smooth muscle.

We investigated the effect of 350 μm halothane on the cADPR induced Ca 2+release. Figure 2demonstrates the effect of halothane on cADPR-induced Ca2+release, addition of 350 μm halothane did not produce any significant Ca2+release by itself; however, it sensitized the Ca2+release system to cADPR (fig. 2). The half-maximal concentration of cADPR was decreased more than fourfold by pretreatment of the microsomes with 350 μm halothane (fig. 2B), although the maximum Ca2+release response to cADPR was not enhanced by halothane. Thus halothane increased the apparent affinity of the Ca2+-induced Ca2+release to stimulation by cADPR. We also observed that 350 μm halothane had no effect steady-state Ca2+levels in the microsomal preparations. The effect of halothane on the cADPR-induced Ca2+release was abolished by the cADPR antagonist 8-Br-cADPR (fig. 3). Furthermore, the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin was not able to potentiate Ca2+induced by agonists of the RyR (data not shown). These observations further support the hypothesis that halothane at the concentration tested sensitizes the RyR.

Fig. 2. Effect of halothane on cADPR-induced Ca2+release in tracheal smooth muscle microsomes. In (A
) Ca2+uptake was initiated by the addition of 1 mm ATP in the presence or not of 0.35 mm halothane after the uptake reached steady state cADPR was added and Ca2+release was monitored with fluo-3 (as described in Materials and Methods). I (B
) dose dependence for cADPR is shown. Ca2+release was induced by different concentrations of cADPR in the absence, or in the presence of 0.35 mm halothane.

Fig. 2. Effect of halothane on cADPR-induced Ca2+release in tracheal smooth muscle microsomes. In (A
) Ca2+uptake was initiated by the addition of 1 mm ATP in the presence or not of 0.35 mm halothane after the uptake reached steady state cADPR was added and Ca2+release was monitored with fluo-3 (as described in Materials and Methods). I (B
) dose dependence for cADPR is shown. Ca2+release was induced by different concentrations of cADPR in the absence, or in the presence of 0.35 mm halothane.

Fig. 3. Effect of 8-Br-cADPR on halothane induced sensitization of the cADPR-induced Ca2+release. Experiments were carried out as described in Materials and Methods. Values are mean ± SD. The Ca2+release induced by 1 μm cADPR was tested in microsomes pretreated with no addition (control), 350 μm halothane (halothane), or 350 μm halothane and 10 μm 8-Br-cADPR (halothane +8-Br-cADPR).

Fig. 3. Effect of 8-Br-cADPR on halothane induced sensitization of the cADPR-induced Ca2+release. Experiments were carried out as described in Materials and Methods. Values are mean ± SD. The Ca2+release induced by 1 μm cADPR was tested in microsomes pretreated with no addition (control), 350 μm halothane (halothane), or 350 μm halothane and 10 μm 8-Br-cADPR (halothane +8-Br-cADPR).

In conclusion, we present evidence that halothane can interact with the new second messenger system modulated by cADPR in tracheal smooth muscle cells. It is possible that the effect of halothane on cADPR may play an important role in the complex effect of volatile anesthetics on intracellular Ca2+homeostasis in these cells. Halothane can promote depletion of the intracellular Ca2+stores by a mechanism that appears to involve leakage of Ca2+through both the IP3and RyR. 6 Our current results indicate that halothane-induced Ca2+leakage may involve sensitization of the RyR to endogenous levels of intracellular cADPR. Increased sensitivity of the RyR to endogenous cADPR induced by halothane may lead to depletion of sarcoplasmic reticulum intravesicular Ca2+levels. This decrease in SR Ca2+will decrease the amount of Ca2+available for SR Ca2+release during agonist stimulation leading to decreased contraction.

Fig. 2. Effect of halothane on cADPR-induced Ca2+release in tracheal smooth muscle microsomes. In (A
) Ca2+uptake was initiated by the addition of 1 mm ATP in the presence or not of 0.35 mm halothane after the uptake reached steady state cADPR was added and Ca2+release was monitored with fluo-3 (as described in Materials and Methods). I (B
) dose dependence for cADPR is shown. Ca2+release was induced by different concentrations of cADPR in the absence, or in the presence of 0.35 mm halothane.

Fig. 2. Effect of halothane on cADPR-induced Ca2+release in tracheal smooth muscle microsomes. In (A
) Ca2+uptake was initiated by the addition of 1 mm ATP in the presence or not of 0.35 mm halothane after the uptake reached steady state cADPR was added and Ca2+release was monitored with fluo-3 (as described in Materials and Methods). I (B
) dose dependence for cADPR is shown. Ca2+release was induced by different concentrations of cADPR in the absence, or in the presence of 0.35 mm halothane.

Fig. 3. Effect of 8-Br-cADPR on halothane induced sensitization of the cADPR-induced Ca2+release. Experiments were carried out as described in Materials and Methods. Values are mean ± SD. The Ca2+release induced by 1 μm cADPR was tested in microsomes pretreated with no addition (control), 350 μm halothane (halothane), or 350 μm halothane and 10 μm 8-Br-cADPR (halothane +8-Br-cADPR).

Fig. 3. Effect of 8-Br-cADPR on halothane induced sensitization of the cADPR-induced Ca2+release. Experiments were carried out as described in Materials and Methods. Values are mean ± SD. The Ca2+release induced by 1 μm cADPR was tested in microsomes pretreated with no addition (control), 350 μm halothane (halothane), or 350 μm halothane and 10 μm 8-Br-cADPR (halothane +8-Br-cADPR).